Biocontrol Mechanisms Of Pseudomonas Fluorescens Strain P-72-10on Black Shank Disease Of Tobacco

Tobacco is an important cash crop. Tobacco black shank, caused by Phytophthora nicotianae Breda de Haan, is one of the most destructive of soil-borne disease in China and other major tobacco-producing areas throughout of the world. The pathogen caused damage to tobacco at all growth stages, with a range from minor injury to complete destruction on a tobacco plant. Currently, this disease is mainly controlled through the use of the fungicide metalaxyl. Nevertheless, increasing concerns about environmental problems caused either directly or indirectly by the use of fungicides and metalaxyl-resistant strains of Phytophthora support the need to find supplemental or alternative control methods such as biological control. Biological control of plant pathogens is an ecologically mind and environmentally safe alternative to chemical control. This study focuses on screening the most effective antagonistic strain against P. nicotianae from five rhizobacteria isolates; the antagonistic mechanisms against pathogen and biocontrol efficiency on black shank disease of this isolate; the colonization dynamics and distribution of this isolate along the roots of tobacco; the influence on the growth of tobacco and its physiological and biochemical mechanisms; the taxonomic position of this isolate and the optimal shake ferment condition of antagonistic compounds produced by this isolate.The detailed results of the study are shown as follows.Screening the most effective antagonistic strain P-72-10Five antagonistic rhizobacteria isolates showed significant suppressive effect on P. nicotianae. Among these isolates, strain P-72-10had the highest suppressive effect, which produced an inhibition zone of13.0mm (radius) and a growth suppression of the fungus at68.57%on the dual culture. It was found in the study that the growth of pathogen's hypha was also suppressed by the extracellular metabolites of strain P-72-10. With the concentrations of extracellular metabolites increasing from10-3to1×, the inhibitory effect enhanced with a percentage from24.92%to46.03%. As compared with the control, the increasing variance was much significant (P<0.05).Biocontrol effect of strain P-72-10on black shank disease When tested on the tobacco plants grown in green house, strain P-72-10reduced disease incidence and declined the disease index significantly. This strain got a biocontrol effect on tobacco black shank disease infection and the effect on the resistant and susceptible varieties of tobacco were53.57%and66.37%respectively.Antagonistic mechanisms against Phytophthora nicotianae of strain P-72-10The volatile metabolites produced by strain P-72-10suppressed the growth of pathogen at32.94%. Strain P-72-10was observed microscopically to cause excessive branching of the fungal mycelia and malformation of mycelia tips; the hypha of pathogen could not grow normally and they formed chlamydospore-like, thick-walled cells with concentrated plasmas. However, the cell wall of normal hypha was even and smooth, and the form was full with the homogeneous and transparent cytoplasm. On the plate culture, strain P-72-10excreted proteinase, celluase, and siderophore, but not chitinase.Colonization dynamics and distribution along tobacco roots of strain P-72-10To study colonization along tobacco roots, strain P-72-10was tagged with antibiotics rifampicin capable of constitutive expression of antibiotics resistance on the King's B medium plate. The rifampicin-tagged bacteria strain colonized successfully on the tobacco roots following seeds inoculation with the rifampicin-tagged bacteria strain. The dynamics of colonization increased first, then decreased and also the considerable quantity of the rifampicin-tagged bacteria strain maintained for a while with an amount level of10cfu/g. The observations by scanning electron microscopy Scanning electron microscopy (SEM) showed that colonization pattern of rifampicin-tagged bacteria strain on the tobacco roots was inhomogeneity. Colonization on the basal part of root reached the maximum, the middle root was less, and then the upper root.Physiological and biochemical influence of strain P-72-10on tobacco seeds and seedlings The root elongation of tobacco was significantly increased for the seed with the treatment of inoculation with strain P-72-10(0.53cm vs0.47cm, P<0.05), and increased by12.76%comparing with the treatment of control. The contents of soluble protein and total amylase in seeds were also increased. Strain P-72-10promoted growth, enhanced photosynthetic rate, increased chlorophyll content and nutrient absorption of tobacco seedlings. MDA content of tobacco seedlings was decreased and defense enzymes related to resistance of plant were induced after treatment with root irrigation, such as PPO, POD, PAL, chitinase and beta-1,3-glucanase. The change trend of different enzymes was inconsistent, and also the activities peak and quantity of enzymes were various with the changement of time.Taxonomic position of strain P-72-10The colonies were cream in color on King's B culture. Cells were gram-negative, rod-shaped [(8.1-16.2)×(1.8-4.8) μm], motile owing to one or several polar flagella and did not form endospores. The growth temperature for strain P-72-10ranged from4to41℃with the optimum at30℃. The results of BIOLOG GN2showed that strain P-72-10belonged to Pseudomonas. The genomic G+C mol content was60.72mol%. Phylogenetic analysis of the16S rDNA gene sequence revealed that P-72-10was the most closely related to Pseudomonas fluorescens, with the sequence similarity of99%. Nucleotide number of the accession was HQ888871. Taxonomically, strain P-72-10was identified as P. fluorescens.Shake flask conditions of strain P-72-10In shake flask conditions, the factors influence strain P-72-10growth, included pH, time and temperature with the optimum pattern at pH7.0,52h and25℃. The culture ingredients were optimized as follows:glucose (23g), ammonium sulphate (20 g), potassium nitrate (5.5g), sodium chloride (0.5g), distilled water (1000mL), pH (7.0).In conclusion, results showed that strain P-72-10had antagonistic activity against P. nicotianae and biological control effect on tobacco black shank as well as plant growth-promoting potential to tobacco and root colonization. Strain P-72-10will also be used as a potential biocontrol agent to reduce the disease incidence and protect the environment.